Orthogonal Crosslinking for Protein Stabilization and Cytosolic Delivery

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Large quantities of proteins can readily be produced from bacteria and, therefore, represent a sustainable source of biological catalysts and therapeutics. With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Qing Lin from the State University of New York at Buffalo to develop a chemical modification to proteins that, when used with light, increases their stability and their ability to enter cells. This chemical biology strategy enables the design of protein-based catalysts with increased resistance to structural disruptions from heat and chemicals. In addition, this new modification enables the development of cell-penetrating small proteins derived from antibodies (called nanobodies) for the diagnosis and treatment of disease. The project provides interdisciplinary research opportunities to undergraduate students, including some from the groups underrepresented in STEM fields, through collaborations with the New York State Collegiate Science and Technology Entry Program.Site-specific incorporation of a 2-aryl-5-carboxytetrazole (ACT) linked amino acid into a protein allows the creation of head-to-tail circular proteins through intramolecular photo-crosslinking with a proximal nucleophilic residue. ACT-based orthogonal inter-strand crosslinking is also explored in this project for the synthesis of nanobodies that display positively charged surfaces to enhance cell-penetration. The crosslinking efficiency and selectivity of the various constructs are optimized through chemical synthesis. The effects of orthogonal crosslinking on protein conformational stability and cytosolic uptake are assessed using appropriate biological assays. These studies are expected to yield new insights into the reactivity of photo-generated reactive species inside proteins, which leads to designed proteins with novel structures and functions.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.